Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway

Cholangiocarcinoma (CCA) is often diagnosed late, leading to incomplete tumor removal, drug resistance and reduced chemotherapy efficacy. Curcumin has the potential for anti-cancer activity through various therapeutic properties and can improve the efficacy of chemotherapy. We aimed to investigate the synergistic effect of a combination of curcumin and gemcitabine against CCA, targeting the LAT2/glutamine pathway. This combination synergistically suppressed proliferation in gemcitabine-resistant CCA cells (KKU-213BGemR). It also resulted in a remarkable degree of CCA cell apoptosis and cell cycle arrest, characterized by a high proportion of cells in the S and G2/M phases. Knockdown of SLC7A8 decreased the expressions of glutaminase and glutamine synthetase, resulting in inhibited cell proliferation and sensitized CCA cells to gemcitabine treatment. Moreover, in vivo experiments showed that a combination curcumin and gemcitabine significantly reduced tumor size, tumor growth rate and LAT2 expression in a gemcitabine-resistant CCA xenograft mouse model. Suppression of tumor progression in an orthotopic CCA hamster model provided strong support for clinical application. In conclusion, curcumin synergistically enhances gemcitabine efficacy against gemcitabine-resistant CCA by induction of apoptosis, partly via inhibiting LAT2/glutamine pathway. This approach may be an alternative strategy for the treatment of gemcitabine-resistant in CCA patients.

www.nature.com/scientificreports/Basin subregion.In particular, O. viverrini is a major public-health problem in Thailand and neighboring countries 4 .
Currently, surgical resection of the tumor is the conventional treatment option, but most CCA patients are diagnosed at an advanced stage and removal of the tumor is often incomplete.Remaining options, such as conventional chemotherapy and radiotherapy, are ineffective due to drug resistance.Thus, new effective chemopreventive and adjuvant therapeutic strategies for CCA are needed 5 .The most popular drug used for chemotherapy of CCA is gemcitabine.However, by the time most CCA patients are diagnosed, the tumor is resistant to chemotherapy 6 .Hence, an alternative treatment that focuses on molecules that confer drug resistance in CCA could have potential application.
Curcumin, (diferuloylmethane), a natural product extracted from the rhizomes of turmeric (Curcuma longa), has anti-oxidant, anti-inflammatory, anti-microbial, anti-fibrotic and anti-cancer activities.Curcumin is useful in the prevention and treatment of various diseases such as cancer, autoimmune diseases, neurological diseases, cardiovascular diseases and diabetes 7,8 .Curcumin exerts its therapeutic effects by suppressing the activation of oncogenic transcription factors, anti-apoptotic proteins, and cell cycle proteins, and can induce the expression of pro-apoptotic proteins 9 .Curcumin is being tested for use as an alternative anti-CCA agent 10,11 .It also has the potential to improve the sensitivity of chemotherapy in drug-resistance models in various cancer types including pancreatic cancer 12 , bladder cancer 13 and colon cancer 14 .
The SLC7A8 gene encodes a neutral amino-acid transporter protein called LAT2 (L-type amino-acid transporter-2).LAT2 plays a critical role for the uptake of neutral amino acids, including glutamine, which are essential nutrients for the growth and proliferation of cancer cells, and are crucial to the biosynthesis of products such as nucleic acids and non-essential amino acids 15 .According to data from Oncomine, LAT2 is upregulated in nine different cancers including breast cancer, colorectal cancer, lymphoma and leukemia 16 .Recent research has shown that LAT2 is upregulated in invasive breast cancer 17 and gemcitabine-resistant pancreatic cancer cells 18 .This protein serves as an oncogenic factor and regulates mammalian target of rapamycin activation in a glutamine-dependent manner, promoting glycolysis and reducing the sensitivity of pancreatic cancer cells to gemcitabine 18 .However, the role of LAT2/glutamine in CCA, especially in the cortex of gemcitabine-resistance has not yet been elucidated.Furthermore, whether anti-CCA activities as well as improvement of gemcitabine efficacy are partly mediated through modulation of LAT2 expression is unknown.
In this study, we aimed to investigate the role of LAT2/glutamine in gemcitabine resistance and the therapeutic potential of curcumin, especially for improvement of gemcitabine treatment using both in vitro and in vivo models of gemcitabine-resistant CCA.The results of the study can inform future clinical application.

Curcumin and gemcitabine suppress cell proliferation and colony formation
Anti-CCA activity focusing on cell proliferation was assessed using the MTT assay and clonogenic assay.Curcumin or gemcitabine, administered alone, decreased proliferation of CCA cells (KKU-055, KKU-100,  The apoptosis analysis of KKU-213B GemR cells revealed significant increases in both early and late apoptosis across treatment groups (curcumin and the combination of curcumin and gemcitabine) compared to the untreated control (Fig. 4a,b).Specifically, curcumin (10 µM) significantly increased early (5.0%, p < 0.05) and late (5.0%, p < 0.01) stages of apoptosis, resulting in an overall apoptosis rate of 10.0% (p < 0.001).The combination treatment significantly boosted early (8.4%, p < 0.001) and late (6.5%, p < 0.0001) stages of apoptosis, culminating in an overall apoptosis rate of 14.9%.Interestingly, treatment with gemcitabine alone (500 nM) did not produce a significant difference from the untreated controls in both early (3.4%) and late (6.5%) stages of apoptosis, with an overall apoptosis rate of 9.9%.These results indicate that curcumin and the combination treatment produced a significant induction of apoptosis (compared to controls), with the combination of curcumin and gemcitabine exhibiting a synergistic effect, leading to the highest levels of both early and late apoptosis.

Overexpression of LAT2 protein in gemcitabine-resistant CCA cells
We identified over 50,000 genes, encompassing both non-coding and coding genes.The volcano plot (Fig. 5a) illustrates the up-regulation and down-regulation of genes, with a specific focus on genes associated with the solute-carrier family, such as SLC7A5, SLC43A1, and SLC7A8.Our findings revealed that SLC43A1 and SLC7A8 were up-regulated in KKU-213B GemR compared to KKU-213B (p < 0.05) (Fig. 5a).Notably, SLC7A8, a gene linked to chemoresistance, exhibited a three-fold increase in expression in KKU-213B GemR compared to KKU-213B (p < 0.001) (Fig. 5b).To establish a more compelling link between SLC7A8 gene and the development of gemcitabine resistance in CCA cells.We compared LAT2 protein (encoded by SLC7A8) expression across three CCA cell lines (KKU-055, KKU-100, and KKU-213B) and one gemcitabine-resistant CCA cell line (KKU-213B GemR ).Interestingly, LAT2 was found to be overexpressed in KKU-213B GemR compared with the other cell lines (Fig. 5c,d).In addition, we determined the effect of the combination of curcumin and gemcitabine on the expression of LAT2, glutaminase (GLS), and glutamine synthetase (GS) proteins.This demonstrated that KKU-213B GemR cells treated with this combination showed a significant suppression in the intensity of LAT2 (p < 0.01), GLS (p < 0.05), and GS (p < 0.05) protein compared to the untreated controls (Fig. 5e-h).
We then performed siRNA-mediated SLC7A8 knockdown experiments in KKU-213B GemR and obtained an impressive knockdown efficiency of 55.62%, with a significant decrease in the expression of LAT2 in the transfected cells compared with non-transfected KKU-213B GemR (Fig. 6a,b).Moreover, our results demonstrated a significant decrease in expression level of GLS protein in KKU-213B GemR siSLC7A8 cells compared to the controls, comprising non-transfected cells (p < 0.05), cells treated with Lipofectamine (p < 0.01) and siNontarget gene (p < 0.05) (Fig. 6c).Additionally, GS exhibited a significant decrease compared to non-transfected cells (p < 0.01), cells treated with Lipofectamine alone (p < 0.01) and siNon-target gene (p < 0.05) (Fig. 6d).KKU-213B GemR siSLC7A8 exhibited other noticeable effects, particularly slower cell growth compared to KKU-213B GemR non-transfected cells.The proliferation of KKU-213B GemR siSLC7A8 cells was significantly lower than non-transfected cells at 48-72 h post-transfection (p < 0.0001) (Fig. 6e,f).Moreover, MTT assays demonstrated  www.nature.com/scientificreports/ that KKU-213B GemR siSLC7A8 exhibited significantly greater sensitivity to gemcitabine than KKU-213B GemR nontransfected cells, indicated by lower cell proliferation.At 48 h, significant differences were observed at doses of 250 μM (p < 0.01), 1000 μM (p < 0.05), 2000 μM (p < 0.001).Similary, at 72 h, significant differences were noted at doses of 1000 μM (p < 0.001), 2000 μM (p < 0.05) (Fig. 6g).Interestingly, KKU213 GemR siSLC7A8 treated with the combination exhibited a significantly greater reduction in cell proliferation compared to KKU-213B GemR non-transfected cells treated with the same combination at both 24 h and 72 h intervals (p < 0.001) (Fig. 6h).These significant results demonstrate the central role of SLC7A8 in gemcitabine resistance of CCA.The observed reduction in cell proliferation and increased sensitivity to gemcitabine after SLC7A8 knockdown in KKU-213B GemR cells, along with the potential reduction in LAT2 when treated with a combination of curcumin and gemcitabine, likely lead to decreased metabolism of glutamine pathway and strongly suggest a direct link between LAT2 and gemcitabine-resistance in cholangiocarcinoma.This insight into the involvement of LAT2 opens new avenues for understanding and potentially overcoming gemcitabine-resistance in CCA.Employing SLC7A8 targeting, along with a combined curcumin and gemcitabine treatment, may be novel approach to treatment of gemcitabine-resistant CCA.

Combination of curcumin and gemcitabine suppresses tumor progression and decreases the level of LAT2, CK19 and HMGB1 in a gemcitabine-resistant CCA xenograft mouse model and in a hamster CCA model
We tested the efficacy of a combination of curcumin and gemcitabine in a xenograft tumor model using KKU-213B GemR cells.Treatment with either gemcitabine or curcumin alone did not result in a significant difference in tumor growth rate compared to the control group.Specifically, the gemcitabine treated group exhibited the highest tumor growth rate at the endpoint, with a rate of 3238%, whereas the curcumin treated group and the control group showed growth rates of 464% and 391%, respectively.Notably, the group receiving the combined treatment demonstrated the lowest tumor growth rate of 265%, significantly lower than seen in the control group (p < 0.01) (Fig. 7a).Immunohistochemical analysis revealed a significant decrease in the LAT2 positive area in groups treated with curcumin alone and in combination with gemcitabine compared to the untreated group (p < 0.0001) (Fig. 7b,c).Notably, in the gemcitabine-treated group, there was fluid accumulation in the www.nature.com/scientificreports/tumor mass, preventing its use for immunohistochemical study.Nevertheless, the combination treatment group exhibited a significantly greater reduction in LAT2 positive area compared to the curcumin treated group, indicating a synergistic effect (p < 0.0001) (Fig. 7c).At the protein level (Fig. 7d-g), the combination of curcumin and gemcitabine significantly suppressed LAT2 expression (p < 0.001) (Fig. 7d,e).However, the intensity of GLS and GS did not show significant differences in any treatment group compared to the untreated group (Fig. 7d,f,g).
In addition, we successfully induced the development of CCA in hamsters, using O. viverrini infection and administration of NDMA, to confirm the efficacy of the combination of curcumin and gemcitabine found in xenograft mouse model.Our results showed remarkable differences in nodule formation between the experimental groups.In the OV + NDMA + Untreated group, multiple white nodules (size 0.3-0.5 cm) were observed.In the curcumin-and gemcitabine-treated groups, 1-2 white nodules (size 0.3-0.4cm and 0.2-0.8cm, respectively) were found.In contrast, the group treated with the combination of curcumin and gemcitabine displayed only small nodules (size less than 0.3 cm) in the livers (Supplementary Fig. S2a).Hematoxylin and eosin staining provided supporting evidence (Supplementary Fig. S2b,c).In the OV + NDMA + Untreated group, the lesions were classed as cholangiocarcinoma (4+, 44.44% of lesions), cholangiofibrosis (2+, 22.22%), chronic inflammatory cells (2+, 22.22%) and cholangiofibroma (1+, 11.11%).In the OV + NDMA + CUR group, the lesions were cholangiofibrosis (2.5+, 38.46%), followed by cholangiocarcinoma (2+, 30.77%), chronic inflammatory cells (1+, 15.38%) and cholangiofibroma (1+, 15.38%).In the OV + NDMA + GEM group the lesions were classed as chronic inflammatory cells (3.5+, 36.48%)following by cholangiofibrosis (3+, 31.58%),cholangiocarcinoma (2+, 21.05%), and cholangiofibroma (1+, 10.53%).Moreover, this group exhibited cholangitis and fluid accumulation correlated with in the xenograft model.The OV + NDMA + GEM + CUR group did not show lesions of cholangiocarcinoma and cholangiofibroma; only cholangiofibrosis (2+, 66.67%) and chronic inflammatory cells (1+, 33.33%) were found.Supportively, the extent of CK19-and HMGB1-positive areas in the CCA control group were significantly higher than in all treatment groups (p < 0.0001).CK19 and HMGB1-positive areas did not differ significantly between the gemcitabine-treated and curcumin-treated groups.Interestingly, the combinationtreated group showed significantly lower CK19 and HMGB1 levels than the curcumin-treated group (p < 0.01 and p < 0.001, respectively) and the gemcitabine-treated group (p < 0.001 and p < 0.01, respectively) (Supplementary Fig. S2d,e).Overall, while neither gemcitabine or curcumin treatment alone significantly affected tumor growth, a combination of the two markedly inhibited tumor growth and attenuated the histopathologic features associated with CCA progression in both hamster and xenograft mouse models.This suggests a potential synergistic effect of curcumin and gemcitabine.

Discussion
Cholangiocarcinoma is a cancer with poor prognosis.Patients usually present at a late stage, leading to high morbidity and mortality, and poor response to drug treatment.Here, we established a drug-resistant CCA cell line (KKU-213B GemR ) to test the therapeutic effect of a combination of curcumin and gemcitabine in CCA both in vivo and in vitro, especially in resistant CCA cell lines.The results showed that the combination of curcumin and gemcitabine was effective in treating resistant CCA cells, in part reducing expression of the LAT2/glutamine pathway, as summarized in Fig. 8.
Previously, gemcitabine-resistant CCA cell lines (using as parents KKU-M139 and KKU-M214) were established using an approach similar to ours.The resultant KKU-M139/GEM and KKU-M214/GEM cells were 25.88-and 62.31-fold more resistant to gemcitabine than their parent cells with low expression of the cell cycle distribution protein that leads to G2/M arrest 20 .Using RBE-R and QBC939-R cells, other researchers have successfully constructed gemcitabine-resistant CCA cell lines after 8 months of induction.They found an increase of IC 50 values from 3.011 µM to 14.44 µM for RBE-R and 4.501 to 18.19 µM for QBC939-R.Cell cycle arrest tended to occur at the S phase in resistant cells 21 .Our gemcitabine-resistant CCA cell line showed 5.50-and 26.22-fold higher resistance to gemcitabine than the parental cells at 40 and 60 cycles after gemcitabine induction, respectively.This could be related to the different doses of gemcitabine treatment (Figs. 1b and 6e).These results suggest that prolonged and repeated exposure of KKU-213B cells to gemcitabine induces greater resistance to the drug in this cell line.
Curcumin inhibits the activation of oncogenic transcription factors, decreasing the level of anti-apoptotic proteins, and promoting the expression of pro-apoptotic proteins 9,10 .Moreover, curcumin enhances the efficacy of gemcitabine treatment in pancreatic cancer by inducing apoptosis of pancreatic cancer cells, downregulating NF-κB-regulated gene products, suppressing angiogenesis and cell migration 22,23 .Our results showed that curcumin suppresses cell proliferation and colony formation of CCA cells by inducing apoptosis and arresting the cell cycle.The combination of curcumin and gemcitabine significantly enhanced apoptosis compared to either treatment alone.Furthermore, curcumin-treated cells exhibited a tendency to accumulate at the G2/M stage in both parental and gemcitabine-resistant CCA cells.Interestingly, the synergistic effect of curcumin and gemcitabine induced accumulation in the S and G2/M phases in our gemcitabine-resistant CCA cells more effectively than either curcumin or gemcitabine treatment alone.The results are consistent with previous findings regarding the effects of curcumin in various types of cancer 12,[24][25][26] .
Amino acid transporters play an essential role in amino-acid transport, tumor progression, and therapy resistance 27 .SLC7A8 is a member of solute carrier family 7 (SLC7), which encodes the LAT2 protein involved in chemoresistance in various cancers 18,28,29 .LAT2 is a transmembrane protein for transportation of neutral amino acids such as glutamine.Our results indicate that SLC7A8 is overexpressed in gemcitabine-resistant CCA cells compared to parental cells.Knockdown with siSLC7A8 had more than 55% efficacy.Furthermore, the results of increased gemcitabine sensitivity and decreased cell growth after knockdown by siSLC7A8 support the involvement of the SLC7A8 in chemoresistance in cholangiocarcinoma.Moreover, LAT2 was suppressed by this combination of curcumin and gemcitabine both in gemcitabine-resistant CCA cell lines and the xenograft mouse model.Supportively, Feng et al. found that overexpression of LAT2 in a pancreatic xenograft mouse model led to reduced sensitivity to gemcitabine, as indicated by a non-significant difference in tumor volume between the gemcitabine-treated group and the control group 18 .Conversely, overexpression of SLC7A8 has been shown to inhibit growth and migration in lung adenocarcinoma 30 and is associated with good prognosis in estrogen receptor-positive breast cancer 17 .Interestingly, according to data from the Cancer Genome Atlas (TCGA), solute carrier (SLC) transporters can have either favorable or unfavorable prognostic value for the different tumor entities 31 .The divergent prognostic impacts may stem from differences in tumor microenvironment, metabolic dependencies, and the genetic or epigenetic context of the cancer.This environment can vary greatly between different types of cancer and even between patients with the same type of cancer 32,33 .
Down-regulation of LAT2 may reduce glutamine uptake of the cancer cells.Glutamine is metabolized by glutaminase (GLS) and glutamine synthetase (GS or GLUL) to glutamate that is involved in amino acid glycolysis, amino acid metabolism, nucleotide metabolism, fatty acid metabolism and the tricarboxylic acid (TCA) cycle, processes associated with tumor progression and the modulation of the tumor microenvironment (TME).Elevated expression of GLS1 correlates with poor prognosis in various cancer types, including CCA, by inducing metastasis and invasion, and promoting the epithelial-mesenchymal transition 34,35 .Similarly, the overexpression of GS, is associated with poor prognosis in various cancer types such as breast cancer 36 , glioblastoma 37 , prostate cancer 38 and hepatocellular carcinoma 39 .Therefore, targeting GLS and GS presents a promising therapeutic strategy.In our result, we found that the expression of GLS and GS decreased significantly after knockdown of KKU-213B GemR with siSLC7A8 and treatment with a combination of curcumin and gemcitabine.This is consistent with previous findings where curcumol and the combination of curcumin and cisplatin attenuated glutaminase activity in hepatic stellate cells and colon cancer cells 14,39,40 .Conversely, in pancreatic cancer cells, GLS levels increased and GS levels decreased after SLC7A8 knockdown 18 .These somewhat contradictory findings may be due to variations in the complex nature of carcinogenesis, the TME and the metabolic demands of each cancer type 19,41,42 .Moreover, our gemcitabine-resistant CCA model was consistent with in vitro results by suppressing tumor growth rate and LAT2 staining intensity.Although GLS did not show significant differences compared to the untreated group, the combination treatment group showed a trend toward decreased GLS.The efficacy of the combination of curcumin and gemcitabine in a gemcitabine-resistant cancer xenograft model was supported in a previous study 12 .
In order to mimic human Opisthorchis viverrini-associated cholangiocarcinoma (CCA), we evaluated the synergistic effects of a combination of curcumin and gemcitabine in an orthotopic hamster CCA model.This model typically originates from infection-induced inflammation and chronic irritation, as documented in previous studies 43,44 .Our results showed that the combination group displayed mainly cholangiofibrosis, a precancerous lesion, without the appearance of tumor foci.In contrast, although cancerous lesions trended to fewer in both the curcumin and gemcitabine groups than in the untreated control group, CCA was still present.The combination group also decreased the intensity of cytokeratin 19 (CK19) and high-mobility group box 1 (HMGB1) staining in liver tissues, markers for confirming the bile-duct epithelial origin of CCA and inflammation-associated carcinogenesis, respectively [45][46][47] , leading to the inhibition of CCA progression in the hamster model.This finding www.nature.com/scientificreports/consistent with our previous results 11,48,49 .A preliminary study in hamster model strongly supports findings in a xenograft model.To achieve a better understanding of the underlying mechanism, larger sample sizes in the hamster CCA model and SLC7A8 knockout in a gemcitabine-resistant xenograft model are required for future studies.
In conclusion, our study shows that the combination of curcumin and gemcitabine synergistically exert an anticancer effect, especially in gemcitabine-resistant CCA cells.Moreover, our in vivo data confirm the efficacy of this combination therapy.Specifically, we identified that SLC7A8 (LAT2) regulates the glutamine mechanism pathway involved in cancer progression.This pathway is a compelling candidate for association with chemotherapy resistance, and our results demonstrate a clear link between LAT2 protein expression and gemcitabine resistance both in vitro and in vivo.These results highlight the potential of the synergistic effects of curcumin and gemcitabine, together with targeting the LAT2/glutamine pathway, as a promising strategy for the treatment of CCA and possibly other cancers.

Establishment and characterization of drug-resistant (KKU-213B GemR )
The gemcitabine-resistant CCA cell line, KKU-213B GemR , was established by growing the parental KKU-213B cell line in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 µM gemcitabine (GEMITA, Fresenius Kabi, India).Cells were maintained under these conditions for 72 h in a humidified incubator.Then the culture medium was replaced with fresh culture medium.After the cells reached 90% confluence, they were sub-passaged and cultured in the presence of 5 µM gemcitabine.After repeating this for 40 cycles, gemcitabine resistance was assessed using the MTT assay (Invitrogen, Thermo Fisher Scientific, MA, USA).In all experiments, gemcitabine induction included up to 60 cycles.

Library preparation and RNA sequencing
Total RNA was extracted from three biological replicates each of KKU-213B and KKU-213B GemR using RNA extraction kit (Invitrogen) following the manufacturer's instruction.The RNA samples were evaluated for degradation and contamination using a 1% agarose gel.Purity of the RNA was determined with the NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA), and RNA integrity and quantity were measured using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).Whole-transcriptome sequencing was performed using paired-end sequencing (2 × 150 bp) on a NovaSeq high-throughput sequencer (Illumina, Inc., San Diego, CA, USA).HISAT2 alignment program (cite: https:// www.nature.com/ artic les/ nmeth.3317) was used for mapping RNA-seq reads to a reference human genome.The read counts were normalized, and differential gene expression analysis performed with R language using the DESeq2 package (version: 1.40.2).The cutoff-value for differential expression analysis was log2 fold-change > 1.0 with p-value < 0.05.Volcano and bar plots were created with ggplot2 (version: 3.4.4).

Measurement of cell proliferation using the MTT assay
CCA cells were seeded at 2000 cells per well in flat-bottomed 96-well plates (Corning).Starting the following day, cells were incubated for 24-72 h with either various concentrations of curcumin (> 97% purity w/w, Merck-Schuchardt, Hohenbrunn, Germany), gemcitabine, or a combination of curcumin and gemcitabine, in a humidified incubator.The culture medium was removed and replaced by 100 µl DMEM without FBS or antibiotic.10 µl of MTT reagent was added to a final concentration of 0.5 mg/mL and the plates were incubated in a humidified incubator for 2 h.The culture medium was then removed and 100 µL of DMSO was added to solubilize the cells.The absorbance at 540 nM was determined using the Varioskan™ microplate reader (Thermo Fisher Scientific, MA, USA).The absorbance of untreated cells was used as a control.The synergistic effect was calculated using SynergyFinder Plus 52 .Half-maximal inhibitory concentration (IC 50 ) was determined using the dose-response inhibition model available in GraphPad Prism 9.0 (GraphPad Software, Inc., CA, USA).

Clonogenic assay
KKU-213 and KKU-213 GemR cells were seeded at 1000 cells per well in 6-well plates and grown overnight in culture medium.Cells were then incubated for 24 h with either curcumin or gemcitabine.After 24 h, the cells were transferred to normal medium and allowed to undergo colony formation for 7 days (KKU-213B) or 12 days (1) Tumor volume = (Width 2 × Length) 2 (2) Tumor growth rate, TRG (%) = (Tumor volume) Initial tumor volume × 100

Figure 1 .
Figure 1.Half-maximal inhibitory concentration (IC 50 ) in CCA cell lines incubated for 48 h or 72 h with the indicated concentration of gemcitabine.(a) Parental CCA cells (KKU-213B); 0-500 nM and (b) Gemcitabineresistant CCA cells; 0-1,000 nM.All data are expressed as mean ± SD from biological and technical replicate experiments.

Figure 2 .
Figure 2. Gemcitabine or curcumin treatment for 48 h and 72 h inhibited cell proliferation (MTT assay) in four CCA cell lines.(a) Gemcitabine; 0-500 nM, (b) curcumin; 0-35 µM, cell proliferation was calculated based on the untreated control of each cell line and expressed as percentage of relative cell viability.(c + d) Representative colony formation assays of CCA cell lines treated with gemcitabine or curcumin.All data are expressed as means ± SD of three independent biological replicate experiments; ns = not significant, # p < 0.0001.G: gemcitabine, C: curcumin.

Figure 3 .
Figure 3.The inhibition of proliferation (MTT assay) by KKU-213B GemR cells after 72 h of incubation with a combination of gemcitabine and curcumin.(a) The combination of gemcitabine (100 nM and 500 nM) and curcumin (5 µM and 10 µM).(b) Percent inhibition and (c) synergy score using the HSA model.All data are expressed as the mean ± SD of biological and technical replicate experiments; ns = not significant, **p < 0.01, ***p < 0.001, and # p < 0.0001 compared between treatment groups.G: gemcitabine, C: curcumin.

Figure 5 .
Figure 5. Overexpression of LAT2 protein in gemcitabine-resistant CCA cells.(a) Volcano plot of differentially expressed genes of KKU-213B (parental) vs. KKU-213B GemR .Green dots indicate downregulated genes, orange dots indicate upregulated genes, and brown dots indicate stable genes.(b) The fold change of SLC7A8 expression in KKU-213B compared to KKU-213B GemR .(c + d) LAT2 expression and intensity in parental CCA cells and gemcitabine-resistant CCA cells and (e-h) LAT2, glutaminase (GLS) and glutamine synthetase (GS) expression and intensity after treatment with gemcitabine, curcumin and the combination of curcumin and gemcitabine.Original blots are presented in Supplementary TableS2.Data are expressed as mean ± SD of three replicate experiments; ns = not significant, *p < 0.01, **p < 0.01 and ***p < 0.001 compared between groups; Log2FC: Log2 fold-change.

Figure 6 .
Figure 6.LAT2 promotes chemotherapeutic sensitivity against gemcitabine-resistant cell.Western blot analyses of (a-d) LAT2, glutaminase (GLS) and glutamine synthetase (GS) in KKU-213B GemR knockdown SLC7A8.(e) Cell morphology and confluence of cells before and after transfection at 48h (10X magnification).(f) Cell proliferation using MTT assay after transfection with siSLC7A8 at 48 h and 72 h (OD 540), treatment with (g) gemcitabine 0-2000 μM at 48 h and 72 h and (h) the combination of curcumin and gemcitabine at 24-72 h.Original blots are presented in Supplementary TableS3.Western blot data are expressed as mean ± SD of biological tripicates and cell proliferation are expressed as mean ± SD of biological and technical replicate experiments; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and # p < 0.0001 compared to control and between groups.G: gemcitabine, C: curcumin.

Figure 7 .
Figure 7. Anti-tumor effects of curcumin, gemcitabine and their combination in gemcitabine-resistant xenograft mouse model.(a) Tumor growth rates and images of tumor tissue (N = 5 in each group), (b) LAT2 immunohistochemical staining in the untreated group, curcumin-and combination-treated groups (the gemcitabine-treated group was not included due to fluid accumulation in the tumor mass) and (c) the positivestaining area (%) of LAT2.(d-g) Protein intensities of LAT2, GLS and GS.Original blots are presented in Supplementary TableS4.All data are expressed as means ± SD of three biological independent experiments; ns = not significant, ** p < 0.01, ***p < 0.001 and # p < 0.0001 compared between groups (N = 3 in each group).CUR: Curcumin, GEM: Gemcitabine.

Figure 8 .
Figure 8.This summary schematic demonstrates that curcumin enhances gemcitabine's ability to (I) suppress the SLC7A8 (LAT2)-regulated glutamine pathway, (II) inhibit GLS, and (III) inhibit GS.This leads to reduced ability of glutamine (Gln) to participate in synthesis of nucleotides, and of glutamate (Glu) being unable to enter the tricarboxylic acid (TCA) cycle and synthesize glutathione and amino acids19 .These mechanisms result in inhibited cell proliferation, induced cell cycle arrest, and apoptosis, ultimately inhibiting cancer progression in gemcitabine-resistant cholangiocarcinoma.